The long-term goal of our program is to establish new genetic strategies that could help elucidate the relevance of mtDNA mutations in mitochondrial dysfunction as they relate to aging. The coexistence of wild-type and mutated mitochondrial genomes (heteroplasmy) is a condition found in mitochondria-related diseases. Due to the stochastic distribution of mtDNA upon mitochondria and cell division, and the accumulation of de novo mutations with the aging of tissue, the percent- age of heteroplasmy among diagnosed patients and even within the tissues of the same patient varies. Better criteria to determine the link between mtDNA mutations and clinical phenotypes could evolve from determining distributions of correlations of a mutation with mitochondrial activity in single mitochondria. Our specific aims include: (1) determining activity with JC-1, mtDNA copy number with SYTO 11, total protein with Mitotracker Green, and membrane size with 10-nonyl acridine orange in single mitochondria from mouse hybrydoma NS-1 cells by capillary electrophoresis with post- column laser-induced fluorescence detection; (2) harvesting separately single mitochondria in micromachined microwells; (3) defining PCR/restriction fragment analysis protocols for detection of the A3243G mutation associated with mt tRNA Leu 1 in normal and photomodified NS-1 cells; (4) performing the molecular biology protocols defined above in a microwell containing a single mitochondrium; (5) detecting and correlating the absence or presence of this mutation with activity determined from a single mitochondrium. Correlated pairs of activity/mtDNA and activity/protein with the chosen mutation for all mitochondria analyzed can define a distribution that could present improved criteria for heteroplasmy. Future expansions to this project will include: use of human cell lines from the NIA Repository that are characterized by mtDNA mutations; application of this genetic strategy to a single cell to study inter- mitochondrial and inter-cellular heteroplasmy; incorporation of biocomputing resources for analysis of Gigabyte size data clusters; use of multiple capillary instruments and development of microfabricated devices for fast heteroplasmic analysis.